The present invention relates generally to tissue implant material for use in grafting procedures. More particularly, the present invention provides non-vascular tissue for use as vascular graft material. The present invention further contemplates a method of vascular grafting using non-vascular tissue. The tissue of the present invention is preferably autologous relative to the recipient of the graft and is conveniently prepared around or on a molding support inserted into a body cavity of the intended recipient of the graft. The tissues and methods of the present invention are particularly useful in the treatment or prophylaxis of diseased or damaged blood vessels such as in atherosclerosis.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Tissue grafting represents a major advance in the medical treatment of diseased or damaged tissue. In some cases, tissue grafting represents the sole avenue of medical treatment. However, the success of tissue grafting depends on a range of factors including the availability of suitable donor tissue and the extent of immunological intolerance by the recipient.
An example of grafting is vascular grafting which is one approach in dealing with atherosclerosis. Atherosclerosis is the principal cause of heart disease, stroke and gangrene of the extremities. Atherosclerotic lesions are a result of an inflammatory response to a damaged artery wall and is associated with excessive lipid deposition (Schwartz et al, 1993). The development of atherosclerosis (atherogenesis) is complex and involves several cell types such as macrophages, T-cells and smooth muscle cells of the intima. Atherosclerosis is responsible for a high rate of mortality and an even higher rate of long term physical impairment of subjects affected by this disease.
A method of treating atherosclerosis is to insert bypass grafts around an artery blocked by plaques. The most common vascular graft material is saphenous vein or mammary artery from the patients. Such graft material is referred to as an autograft. Vein and artery autografts are flexible, viable, non-thrombogenic and compatible. However, while the mammary artery seldom develops atherosclerosis, it may not always be the proper size or length, and saphenous vein may have varicose degenerative alterations that can lead to aneurysm formation when transplanted to a high pressure arterial site. Furthermore, the non-thrombogenic surface of endothelial cells of saphenous veins is often damaged during graft preparation.
Similarly, the use of dialdehyde starch tanned bovine xenografts has been generally abandoned due to a high incidence of aneurysm formation and poor resistance to infection.
For these reasons and because autologous grafts not always available, attempts have been made to produce synthetic vascular prostheses. The first synthetic vascular prosthesis was made of Vinyon-N and was implanted into a patient in the late 1940""s. The patient died 30 minutes after the operation. Replacements have been made with nylon, then later with TEFLON which is polytetrafluoroethylene manufactured by DUPONT and DACRON which is a long-chainpolyester made from ethyleneglycol and tesephthalic acid manufactured by DUPONT. Nylon was found to lose most of its tensile strength after a brief period of implantation leading to aneurysmal dilation and graft rupture. Although both DACRON and TEFLON fabric grafts perform reasonably satisfactorily in high flow, low resistance conditions such as in the aorta, iliac and proximal femoral arteries, neither of these two materials is satisfactory for small caliber arterial reconstructions. Such grafts are compounded by graft failures from stenosis at the anastomic sites and excessive intimal hyperplasia. These complications are associated with graft thrombogenicity, poor healing and lack of compliance.
In the early 1970""s, non textile vascular grafts prepared from expanded polytetrafluoroethylene (ePTFE) were introduced. ePTFE is the most chemically inert of all polymeric materials and is not degraded or changed in the chemical environment of the body and is extremely easy to suture. However, poor healing characteristics and lack of compliance are major causes for its lack of performance.
Indeed, the major problem with all synthetic vascular prostheses is that they are foreign bodies, so that blood coagulation can occur on their luminal surfaces causing occlusion in prostheses. One innovation designed to improve the patency of the synthetic vascular graft is to coat the lumen of the vascular graft with endothelial cells. While flow through the graft is improved and thrombogenesis reduced, graft failure can still occur due to occlusion by overgrowth of endothelial cells. In an attempt to control the growth, gene therapy has been used. This refinement addresses the overgrowth, but retrovirally transduced cells on the graft are not able to withstand the shear stresses encountered by flow of blood and are sheared off. Also, the procedure for obtaining endothelial cells from the patient is invasive and the cells are hard to propagate in vitro.
Tissue-polymer prostheses are available which incorporate a combination of tissue and synthetic material in the form of an integral composite. In one form, silicone mandrels covered with DACRON mesh are implanted beneath the cutaneous trunci muscles of sheep where they become encapsulated with ovine collagen (Koch et al, 1997). The tubes are then excised and trimmed of excess fat and connective tissue is then fixed with glutaraldehyde. The silicone mandrel is then removed leaving the fibre-reinforced tube which, after sterilization, is stored in ethanol (Edwards and Roberts, 1992). Although this prosthetic device has been successfully used, it does suffer the disadvantage of lacking elastin, an important component to prevent aneurysmal and dilatory changes from stretching both the collagen and mesh components. Further-more, the prosthetic device uses glutaraldehyde and this has the propensity to induce non-specific calcification of the implanted device.
In summary, despite considerable experimental and clinical research, none of the biological and synthetic grafts produced thus far is an ideal substitute for a blood vessel such as an artery, arterio-venous shunt or an access fistula. Limited availability, graft deterioration and complications such as thrombosis, aneurysm formation and excessive subintimal hyperplasia at the anastomotic sites are major problems.
There is a need, therefore, to develop tissue for use in vascular grafting which exhibits the biocompatibility of a recipient""s own tissue but which is created artificially obviating the need to sacrifice existing, i.e. indigenous, tissue from the recipient. In accordance with the present invention, the inventors have identified a means of producing living graft issue for use as vascular tissue but which is derived from non-vascular tissue.
The present invention is predicated in part on the surprising observation that granulation tissue produced in a cavity of a live body in response to foreign material is useful as grafting material. The granulation tissue comprises non-thrombogenic, mesothelial (endothelial-like) cells overlying several layers of myofibroblasts which, in a preferred embodiment, is highly contractile, strong and responds to agonists and antagonists in a manner similar to smooth muscle in blood vessels. After grafting, elastic fibres are produced by the myofibroblasts.
Accordingly, one aspect of the present invention provides isolated tissue suitable for use in a vascular graft said tissue comprising granulation tissue produced on a molding support, and wherein the tissue is removed from the molding support prior to use.
Although this aspect of the present invention is directed to tissue suitable for use in vascular grafting, the present invention extends to the use of the non-vascular granulation tissue formed in a body cavity in any suitable graft. In a particularly preferred embodiment, the present invention provides living non-vascular tissue for grafting of substitute blood vessels.
Accordingly, another aspect of the present invention provides an isolated substitute blood vessel or a portion thereof comprising granulation tissue covered by non-thrombogenic mesothelial cells wherein the tissue forms on a molding support inserted into a body cavity of the intended recipient of the substituted blood vessel and wherein the tissue is removed from the molding support prior to use.
In one embodiment, the substitute vessel is a substitute for an artery.
In another embodiment, the substitute vessel is a substitute for an arterio-venous shunt or an access fistula.
Reference herein to xe2x80x9cprior to usexe2x80x9d means that prior to the tissue being used in a vascular graft, such as a substitute blood vessel, it is removed from the molding support. This may occur immediately prior to grafting or a period of time before grafting.
In a particularly preferred embodiment, the substitute artery is prepared in vivo by inserting a molding support in the form of a tube into a cavity of a live body and maintaining the molding support in vivo until such time as granulation tissue forms on and around the molding. The granulation tissue takes the form of the shape of the molding. The molding support may, in fact, become encapsulated. Preferably, therefore, where the tissue is for use as a substitute artery, the molding is a hollow or solid tube with a desired length and diameter. The molding needs to provoke an inflammatory response. In this regard, in a preferred embodiment, the molding support is recognised by the recipient as a foreign body. The molding support may or may not need to be sterile.
Although not wishing to limit the present invention to any one theory or mode of action, it is proposed that peritoneal or other body cavity macrophages coat the molding support together with other cells of the immune system such as but not limited to cells involved in an immune-mediated inflammatory response. Cells proposed to be involved include granulocytes, macrophages and stromal cells. The macrophages eventually take on a flattened appearance, and fibroblasts cells as well as cells with an intermediate morphology appear. Eventually, a continuous layer of mesothelial cells and cells resembling myofibroblasts forms. The cytoplasm of these cells shows the abundant rough endoplasmic reticulum seen in normal fibroblasts but also contains massive but discrete bundles of microfilaments with dense bodies which closely resemble those of smooth muscle cells. This tissue is referred to herein as xe2x80x9cgranulation tissuexe2x80x9d. The molding support is then removed from the body cavity and separated from the cells and discarded. It may be necessary in order to separate the cells from the molding support to cut or sever parts or portions of the tissue. In the case of the preparation of a substitute artery, the molding is in the form of a tube and the tissue remaining after the tube is discarded is everted, i.e. turned inside out, such that the mesothelium lining the granulation tissue is now lining the inside of the substitute vessel thus mimicking the structure of a normal blood vessel. In this regard, the tissue generally encapsulates the tubular molding and needs to be cut at one or both ends in order for the molding to be separated from the tissue.
Accordingly, another aspect of the present invention provides an isolated substitute blood vessel or a portion thereof comprising a tubular tissue section comprising living myofibroblasts within granulation tissue wherein the tissue is formed on a tubular mold and then removed from the mold and everted.
Yet another aspect of the present invention provides an isolated tissue suitable for use in a vascular graft said tissue produced by the process of placing a molding support within a body cavity for a time and under conditions sufficient for granulation tissue to form on said molding support, removing the molding support from the cavity and then removing the tissue from the molding support and everting the tissue.
Still yet another aspect of the present invention contemplates a method of producing substitute tissue, said method comprising placing a molding support within a body cavity for a time and under conditions sufficient for granulation tissue comprising myofibroblasts to form, removing said molding support from the body cavity and separating the granulation tissue from said molding support.
In a particularly preferred embodiment, the present invention is directed to a method for producing a substitute blood vessel said method comprising inserting into a body cavity a molding support in the form of a tube for a time and under conditions for granulation tissue with myofibroblasts to form, removing the tubular molding from the body cavity, separating the tubular molding away from the granulation tissue and everting said granulation tissue.
Any body cavity may be used including but not limited to the peritoneum, thoracic cavity, scrotum, brain, joint or pericardial cavity. Preferably, the cavity is lined with mesothelial cells. The peritoneal cavity is the most convenient and least disruptive to the host and is preferred in accordance with the present invention.
The molding support may be surgically implanted into the body cavity where it is effectively placed without restraint in the cavity.
Alternatively, the molding support is fixed to a region within the cavity. This may make insertion and/or retrieval of the implant easier. For example, a molding support may be provided by way of a catheter. In this regard, a molding support such as a tubular molding can be provided to the peritoneal cavity, for example, via a prosthetic device such as a peritoneal dialysis catheter. One example of a peritoneal dialysis catheter is a Tenckhoff catheter. This provides a convenient manner in which to gain access to the molding in the peritoneum by a less invasive procedure than open surgical intervention. A catheter may be employed as a source of tubular molding per se, i.e. that piece of the catheter inserted into the cavity or the catheter may be used as a conduit for passing suitable molding supports into and out of the cavity.
Accordingly, another aspect of the present invention provides a prosthetic device which facilitates the provision of a molding support to a body cavity, said prosthetic device comprising an elongated member, said member having a portion adapted to be inserted into a body cavity and a portion adapted to be external to the body cavity wherein the portion adapted to be inside the body cavity comprises a molding support or permits entry of a molding support into said body cavity wherein granulation tissue forms on or around said molding support which granulation tissue is suitable for use as a vascular graft, such as substitute blood vessel.
In accordance with this embodiment, the internal portion of the elongated member of the catheter may be the molding support per se. Alternatively, the elongated member may be a hollow tube through which a molding support may be passed from the portion of the catheter external to the cavity to the portion of the catheter in the cavity. In the case of the latter embodiment, the molding support would preferably be extended past the terminal portion of the portion inside the cavity such that the molding support or part thereof is exposed to the cavity. Conveniently, a line or wire or other means is attached to one part of the molding support to facilitate retrieval of the molding support through the elongated member.
The portion of the member external to the body cavity may still be located inside the body but outside the lining of the body cavity. For example, the external portion may be positioned subcutaneously. Alternatively, the external portion is outside the body.
Preferably, the body cavity is the peritoneal cavity.
Preferably, the elongated member is a filament or tubular mold.
Another aspect of the present invention provides a filament or tubular mold support capable of acting as a catheter for a body cavity wherein one portion of said filament or tubular mold support is present in the body cavity and another portion of filament or tubular mold support is outside the body cavity.
In one embodiment, the prosthetic device or filament or tube is packaged for sale with instructions for use.
In one preferred embodiment, a Tenckhoff catheter or its functional equivalent is used. This may have a single or double cuff of DACRON to prevent migration of bacteria and, hence, peritionitis when used in the peritoneal cavity, and may be used with or without silicon discs to hold the omentum and bowel away from the tubing. Conveniently catheters are inserted into the peritoneal cavity over a guide wire through an incision, generally after first infusing with dextrose dialysis solution. The cuff is then sewn in place in the peritoneum and an adapter attached to the external portion of the catheter.
The present invention is particularly directed to the use of body cavities to prepare the substitute tissue. This is done, however, with the understanding that the present invention extends to preparing substitute tissue in vitro. For example, through tissue culture techniques including feeder layers, granulation tissue may be induced to form on or around a molding support. The use of in vitro culture techniques has an advantage in that culture conditions can be manipulated and controlled such as by the addition of, for example, growth factors and cytokines. It also has the advantage of not requiring an invasive procedure in order to produce the artificial artery. Generally, an artificial vessel is made in vitro with no artificial support scaffold but with a scaffold of matrix it has created itself, as with the mesothial-lined granulation tissue tube formed in a body cavity of a host. The production of artificial vessels in vivo and in vitro both have advantages and both techniques are contemplated by the present invention.
The molding support is selected depending on the intended use of the tissue. For example, tubes, beads or discs may be used. Tubes are used for the preparation of substitute blood vessels. Discs and beads may be used for repairing internal organ or tissue damage.
The molding support may be any material including polymers such as cellulose, polyacrylamide, nylon TEFLON, DACRON, polystyrene, polyvinyl chloride, polypropylene, silastic tubing and polytetrafluoroethylene. The use of glass is also contemplated by the present invention but is not a preferred molding support. Reference to xe2x80x9ctubular moldingxe2x80x9d is not to be taken as limiting the molding to a hollow tube. The present invention also contemplates a molding support in the form of a filament such as a solid fibre. Up to the present time, plastic silastic tubing is the most useful form of molding support for the preparation of substitute blood vessels.
In a particularly preferred embodiment, the present invention contemplates a method for producing a substitute blood vessel said method comprising inserting a molding support in tubular form into the peritoneal cavity of a recipient for a time and under conditions sufficient for granulation tissue to form with myofibroblasts, removing the molding from the peritoneal cavity, separating the molding away from the granulation tissue and everting said granulation tissue.
Preferably, the molding support is silastic tubing or its equivalent. The length and diameter of the substitute blood vessel is determined by the length and diameter of the tubing employed as the molding support. Conveniently, the diameter of the tubing may range from about 0.1 mm to about 10 mm and more preferably from about 0.5 mm to about 5 mm. The length of the tubing will depend on the amount of graft required and on the size of the body cavity. For example, a length of from about 0.1 mm to about 1000 mm, more particularly from about 1 mm to about 800 mm and even more particularly from about 3 mm to about 500 mm may be employed. More preferably, the length is from about 10 mm to about 250 mm. In addition, this procedure permits branched or looped tubing being employed to generated branched or looped blood vessel grafts.
The method of the present invention is particularly useful for generating substitute blood vessels since the substitute blood vessels of the present invention exhibit a non-thrombogenic surface, have compliance and elasticity, exhibit long-term tensile strength, are biocompatible, are easy to handle and have suturability and are available in any size depending on the size and shape of the tubular molding. The tubular molding may also comprise spiral grooves. The spiral orientation of smooth muscle cells in blood vessels facilitates control of compliance.
The preferred recipient for the implantation of the molding support is the patient requiring the substitute blood vessel or transplant. However, it is within the scope of the present invention for substitute blood vessels or other transplantable tissue to be prepared in other individuals such as genetically related individuals or non-related individuals. In the case of the latter, immune-suppressing therapy may be required to effect the transplant.
The present invention is particularly directed to grafting in humans although the subject invention extends to other animals and birds such as primates, laboratory test animals (e.g. mice, rats, rabbits, guinea pigs), livestock animals (e.g. cows, sheep, pigs, horses, donkeys), companion animals (e.g. dogs, cats), captive wild animals, caged birds, game birds and poultry birds (e.g. chickens, geese, ducks, turkeys).
The present invention further contemplates the genetic manipulation of the substitute tissue. In one embodiment, once the tissue is removed from the body cavity, the mesothelial cells are transfected with a viral vector, naked DNA or other suitable genetic vehicle. Alternatively, or in addition, the myofibroblasts may be genetically manipulated. Generally, the aim of genetic manipulation is to introduce traits which facilitates function or operation of the graft. For example, genes encoding tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA) or streptokinase may be introduced. Alternatively, or in addition, genes may be introduced such as which encode nitric oxide synthase (NOS) which prevents unwanted clotting and spasm.
The present invention further contemplates a method of treating atherosclerosis or other blood vessel disease said method comprising by-passing or replacing the damaged blood vessel by grafting a substitute blood vessel, said substitute tissue comprising myofibroblasts within granulation tissue.
Preferably, the substitute blood vessel is prepared by placing a molding support comprising a tube in a cavity of a live body, such as a peritoneal cavity, for a time and under conditions sufficient for granulation tissue comprising myofibroblasts covered by mesothelium to form, removing said molding from the body cavity and separating the molding away from the granulation tissue and then everting the granulation tissue.
Yet another aspect of the present invention provides an isolated tissue suitable for use in a vascular graft said tissue produced by the process of placing a molding support within a body cavity for a time and under conditions sufficient for granulation tissue to form on or around said molding support and wherein the tissue is removed from the molding support prior to use.
Preferably, the tissue is suitable for use as a substitute blood vessel or a portion thereof, in which case the molding support is in tubular form.
Preferably, the granulation tissue is covered by non-thrombogenic mesothelial cells. The granulation tissue generally comprises living myofibroblasts within granulation tissue. The living myofibroblasts produce elastic fibres within a few weeks of transplantation to a high pressure arterial site. Elasticity is important to prevent aneurysmal and dilatory changes.
The present invention further provides an isolated substitute blood vessel maintained in a frozen state for use by a mammal in which it is produced said substitute blood vessel formed by placing a tubular molding within a body cavity of said mammal for a time and under conditions sufficient for granulation tissue comprising myofibroblasts to form, removing said tubular molding and separate granulation tissue away from the tubular molding and then everting the granulation tissue.
Preferably, the body cavity is the peritoneal cavity.
Still another aspect of the present invention contemplates the use of a molding support in the manufacture of tissue suitable for use in a vascular graft, said tissue comprising granulation tissue produced on said molding support.
The present invention is now described with respect to the practice of one particular preferred embodiment. The following description is in no way intended to limit the scope of the instant invention.
To prepare a substitute blood vessel, an approximately 20 to 100 mm long piece of approximately 1 to 10 mm diameter silastic tubing comprising spiral grooves is optionally coated with fibronectin which enhances macrophage adhesiveness. The tube is placed in the peritoneal cavity and 10 to 15 ml of balanced salt solution and dextrose added together with a growth factor or cytokine such as but not limited to granulocyte-macrophage colony-stimulating factor (GM-CSF) in order to stimulate macrophage recruitment and proliferation.
The peritoneal cavity is closed and in approximately 1 to 6 weeks and more preferably 2 to 3 weeks later, the tube is removed from the cavity. It is necessary to remove the tissue from the tube by a process of eversion. Generally, the ends of the tissue are cut, the tubing removed and discarded and one end of the tissue held by five forceps inserted through the lumen. The tissue is pulled back through the lumen, completely turning inside out. Generally, heparin is then infused through the everted tissue at the time of grafting into a blood vessel.
The present invention is further described by the following non-limiting Figures and Examples.